V(D)J recombination lies at the heart of antigen receptor diversity and adaptive immunity. The RAG complex (RAG), which includes RAG1, RAG2 and HMGB1, initiates this critical process by binding recombination signal sequences (RSSs) and creating DNA double-stranded breaks (DSBs). The resulting breaks are repaired via the non-homologous end-joining (NHEJ) pathway, the predominant DSB repair mechanism in mammalian cells. Mutations in RAG or NHEJ proteins cause defects in V(D)J recombination leading to joining errors, chromosomal deletions and translocations, and genome instability. Defective V(D)J recombination is associated with a range of human disorders including cancer, common immune deficiency (CID) and severe combined immunodeficiency (SCID), and ionizing radiation (IR) sensitivity. Despite much progress in the field, a particularly critical step of V(D)J recombination?the transition from RAG-mediated DNA cleavage to NHEJ-mediated DNA repair?remains poorly understood. Two particularly glaring gaps in our knowledge of this process are: 1) What are the steps and RAG-NHEJ factor interactions that mediate this process? and 2) How are the RAG and NHEJ complexes organized and regulated (dysregulated) in the ?recombination centers? within which V(D)J recombination takes place in vivo? Research into these questions has been hampered by limitations inherent in traditional biochemical, structural, and cell biological approaches, limitations that can now be overcome by high-resolution single molecule methods. In this application, we propose to address these knowledge gaps by defining the molecular mechanism of the RAG-NHEJ handoff process and how its dysfunction leads to aberrant V(D)J recombination. To accomplish this, we will use of an array of innovative single-molecule techniques and assays. The proposed studies are supported by key preliminary experiments including the application of single-molecule assays to monitor the RAG-NHEJ handoff process in vitro in real-time, and utilization of super-resolution imaging of recombination complexes during transactions of V(D)J recombination in cells.